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Target of rapamycin (TOR) signaling is an essential nutrient-dependent pathway controlling cell growth in all eukaryotes. TOR signaling is well characterized in yeast and animals but remains poorly investigated in plants. The hormonal action of gibberellic acid (GA) is a crucial factor for wheat germination by inducing the synthesis of α-amylase in wheat aleurone cells. Here we showed that GA promotes the activation of Triticum aestivum TOR (TaTOR) signaling as evidenced by increased phosphorylation of T. aestivum S6K1 (TaS6K1) on its conserved hydrophobic motif together with proteasomal degradation of growth-inhibitory factor Rht-1. GA-dependent activation of TaTOR signaling led to α-amylase synthesis and Rht-1 proteasomal degradation because both GA-dependent events were sensitive to TaTOR inhibition. Using antibodies specific to TaTOR, we successfully identified the presence of endogenous TaTOR protein in terminally differentiated wheat aleurone layers. Additionally, by examining the rapamycin-sensitive phosphorylation of S6K1 as a reliable indicator of endogenous TOR kinase activity, we demonstrated that the activity of TaTOR in aleurone layers is enhanced by GA. Importantly, this stimulation is not associated with the regulation of either TaTOR transcription or the accumulation of TaTOR protein. In yeast and pull-down assays, a robust interaction between TaS6K1 and the N terminus of Rht-1 (amino acids 1–234) was observed, a finding further supported by co-immunoprecipitation of endogenous Rht-1 and TaS6K1. Furthermore, the administration of mTOR inhibitors significantly attenuated GA-induced degradation of endogenous Rht-1 and prolonged the persistence of the complex formed by these two proteins. We propose that TaTOR-TaS6K1 signaling contributes to GA-dependent wheat germination by mediating α-amylase synthesis and controlling proteasomal degradation of Rht-1 in wheat aleurone cells.

Acipenser baerii (Siberian sturgeon) is native to Kazakhstan and is currently endangered and is listed within the first class of protected animals. Sturgeon aquaculture is becoming an important tool for the recovery of this endangered species. Nonetheless, diseases involving typical symptoms of skin ulceration and systemic bacterial hemorrhagic septicemia have occurred in cultured A. baerii on a fish farm located in Western Kazakhstan. In this study, an infectious strain of bacteria isolated from an ulcer of diseased A. baerii was identified as Aeromonas salmonicida (strain AB001). This identification involved analyses of 16S rRNA, gyrB, rpoD, and flaA genes’ sequences. Even though strain AB001 belongs to A. salmonicida, it exhibited noticeable mobility and growth at temperatures of ≥37 °C. Profiling of virulence genes uncovered the presence of seven such genes related to pathogenicity. Antibiotic sensitivity testing showed that the strain is sensitive to aminoglycosides, amphenicols, nitrofurans, quinolones, and tetracyclines. Half-lethal doses (LD50) of strain AB001 for Oreochromis mossambicus and A. baerii were determined: respectively 1.7 × 108 and 7.2 × 107 colony-forming units per mL. The experimentally induced infection revealed that strain AB001 causes considerable histological lesions in O. mossambicus, including tissue degeneration, necrosis, and hemorrhages of varied severity.

The Siberian sturgeon (Acipenser baerii) is a long-lived and late-maturing fish; its natural populations are considered endangered and listed in the first class of protected animals in Kazakhstan. To date, attempts have been made to increase the number of sturgeons through artificial reproduction in aquaculture and a release of the resultant juveniles into the Ural River, which flows into the Caspian Sea. This species faces an increased risk of Aeromonas infections. Aeromonas hydrophila is the most important sturgeon pathogen in Kazakhstan, but studies on A. hydrophila infection in Kazakhstan are still inconclusive. In the present work, our purpose was to isolate and characterize dominant bacteria in diseased A. baerii. This isolate, tentatively named AB005, was identified as A. hydrophila in an analysis of its morphological, physiological, and biochemical features and 16S ribosomal-RNA and gyrB gene sequences. A pathogenicity test was carried out for the isolate on healthy Oreochromis niloticus and Acipenser ruthenus via intraperitoneal injection along the caudal peduncle of the fish. Half-lethal doses (LD50) of isolate AB005 for O. niloticus and A. ruthenus were determined: 8.37 × 105 and 2.89 × 106 colony-forming units per milliliter, respectively. Virulence gene profiling revealed the presence of seven virulence genes related to pathogenicity (acyltransferase, phospholipase A, serine protease, heat-stable cytotonic enterotoxin, nuclease, and aerolysins A and B) in this A. hydrophila isolate. Drug sensitivity testing showed that the isolate is sensitive to quinolones, aminoglycosides, nitrofurans, amphenicols, and tetracyclines. The present findings will lay the foundation for future research on this pathogen in Siberian-sturgeon aquaculture.

Abstract Background Reduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein. Results To generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A–agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid. Conclusion The purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

Germination is a process of seed sprouting that facilitates embryo growth. The breakdown of reserved starch in the endosperm into simple sugars is essential for seed germination and subsequent seedling growth. At the early stage of germination, gibberellic acid (GA) activates transcription factor GAMYB to promote de novo synthesis of isoforms of α-amylase in the aleurone layer and scutellar epithelium of the embryo. Here, we demonstrate that wheat germination is regulated by plant target of rapamycin (TOR) signaling. TOR is a central component of the essential-nutrient–dependent pathway controlling cell growth in all eukaryotes. It is known that rapamycin, a highly specific allosteric inhibitor of TOR, is effective in yeast and animal cells but ineffective in most of higher plants likely owing to structural differences in ubiquitous rapamycin receptor FKBP12. The action of rapamycin on wheat growth has not been studied. Our data show that rapamycin inhibits germination of wheat seeds and of their isolated embryos in a dose-dependent manner. The involvement of Triticum aestivum TOR (TaTOR) in wheat germination was consistent with the suppression of wheat embryo growth by specific inhibitors of the TOR kinase: pp242 or torin1. Rapamycin or torin1 interfered with GA function in germination because of a potent inhibitory effect on α-amylase and GAMYB gene expression. The TOR inhibitors selectively targeted the GA-dependent gene expression, whereas expression of the abscisic acid-dependent ABI5 gene was not affected by either rapamycin or torin1. To determine whether the TaTOR kinase activation takes place during wheat germination, we examined phosphorylation of a ribosomal protein, T. aestivum S6 kinase 1 (TaS6K1; a substrate of TOR). The phosphorylation of serine 467 (S467) in a hydrophobic motif on TaS6K1 was induced in a process of germination triggered by GA. Moreover, the germination-induced phosphorylation of TaS6K1 on S467 was dependent on TaTOR and was inhibited by rapamycin or torin1. Besides, a gibberellin biosynthesis inhibitor (paclobutrazol; PBZ) blocked not only α-amylase gene expression but also TaS6K1 phosphorylation in wheat embryos. Thus, a hormonal action of GA turns on the synthesis of α-amylase in wheat germination via activation of the TaTOR–S6K1 signaling pathway.